Biochemical and molecular biological analysis of chromatin domains is critical for understanding of molecular mechanisms of epigenetic regulation. However, the biochemical characteristics of chromatin domains are poorly understood. This is because sampling methods applicable to biochemical and molecular biological analysis of chromatin domains are limited.
The biochemical and molecular biological analysis of chromatin domains requires a sample retaining interaction of genomic DNA and its interacting molecule(s). As a method for isolating a specific genomic region while maintaining interaction of the specific genomic region with its interacting molecule(s), the following examples are known.    (1) Chromatin Immunoprecipitation
Chromatin immunoprecipitation (hereinafter referred to as “ChIP”) is a method involving immunoprecipitation of a specific genomic region with an antibody against a known DNA-binding protein for isolation of the region concerned (see Non Patent Literature 1 and 2). Therefore, there is limitation that ChIP cannot be used without the data regarding DNA-binding proteins. In addition, since DNA-binding proteins generally bind to plural regions of genomic DNA and the resulting immunoprecipitates contain various genomic regions, ChIP has difficulty in isolation of a single specific genomic region. Further, ChIP does not enable isolation of a genomic region to which known DNA-binding proteins do not bind.    (2) Chromosome Conformation Capture (Hereinafter Referred to as “3C”)
3C or its derivatives can be used for identification of a genomic region(s) interacting with a specific genomic region(s) (see Non Patent Literature 3 to 5). However, 3C involves an enzymatic reaction with restriction enzymes or ligases under non-optimal conditions, namely crosslinking conditions, and therefore has a high possibility of detecting unphysiological interactions. In addition, since restriction enzyme digestion is likely to be incomplete under crosslinking conditions, amplification of neighboring regions of the target genomic region is unavoidable in PCR, resulting in an extremely high background, which makes it difficult to detect unidentified interactions.
(3) Fluorescence In Situ Hybridization (Hereinafter Referred to as “FISH”)
FISH alone or in combination with immunofluorescence can be used to detect interaction of a specific genomic region with other genomic regions, RNA and proteins. However, this method has a low resolution and cannot be used for detection of interaction with unidentified molecules.    (4) Proteomics of Isolated Chromatin Segments (Hereinafter Referred to as “PICh”)
PICh utilizes a specific nucleic acid probe for isolation of a specific genomic region, and it is reported that this method enables isolation of telomeric regions having multiple repeats of a sequence complementary to the probe (see Non Patent Literature 6). Regarding PICh, which involves annealing of a nucleic acid probe and a target genomic region under crosslinking conditions, it is not reported whether a low copy number or one copy of a specific genomic region can be isolated.    (5) Method of Non Patent Literature 7
In Non Patent Literature 7, an attempt to isolate a specific genomic region by affinity purification is described. In this attempt, the cell used is yeast and application to higher eukaryotic cells is yet to be tested. In this method, the specific genomic region is cut out with the aid of the Cre-loxP system, and this procedure may alter chromatin structures. In addition, due to lack of crosslinking treatment with formaldehyde, molecules bound to the specific genomic region, such as proteins and DNA, may dissociate in a purification process.
[Citation List]
[Non Patent Literature]
Non Patent Literature 1:
    Solomon, M. J., Larsen, P. L., Varshaysky, A., Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly transcribed gene. Cell (1988) 53, 937-947Non Patent Literature 2:    Solomon, M. J., Varshaysky, A., Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA (1985) 82, 6470-6474Non Patent Literature 3:    Dekker, J., Rippe, K., Dekker, M., and Kleckner, N. Capturing chromosome conformation. Science (2002) 295, 1306-1311Non Patent Literature 4:    Simonis, M., Klous, P., Splinter, E., Moshkin, Y., Willemsen, R., de Wit, E., van Steensel, B., and de Laat, W. Nuclear organization of active and inactive chromatin domains uncovered by chromosome conformation capture-on-chip (4C). Nat. Genet. (2006) 38, 1348-1354Non Patent Literature 5:    Simonis, M., Kooren, J., and de Laat, W. An evaluation of 3C-based methods to capture DNA interactions. Nat. Methods (2007) 4, 895-901Non Patent Literature 6:    Dejardin, J., and Kingston, R. E. Purification of proteins associated with specific genomic loci. Cell (2009) 136, 175-186Non Patent Literature 7:    Griesenbeck, J., Boeger, H., Strattan, J. S., and Kornberg, R. D. Affinity purification of specific chromatin segments from chromosomal loci in yeast. Mol. Cell Biol. (2003) 23, 9275-9282